Regulation Of GTP-binding Proteins

ADP核糖化因子(ARF)是一种GTP结合蛋白，调节许多囊泡运输途径。ARF功能需要GTP结合的活性形式和GDP结合的非活性形式之间的调节交替。GTP结合是由鸟嘌呤核苷酸交换蛋白(GEP)催化的，其中一些GEP被布雷菲尔丁A(BFA)抑制，BFA是一种抑制蛋白质分泌并导致高尔基体池可逆性解体的药物。该小组早期纯化和克隆的两个BFA抑制的GEP(BIG1和BIG2)经常从具有特定抗体的培养细胞中沉淀在一起，但它们在酵母双杂交筛选中的表现截然不同，其中BIG1与FKBP13相互作用，BIG2与环磷酸腺苷活化蛋白激酶(PKA)的调节亚基RIpha相互作用。FKBP13是FK506结合蛋白家族中的一种免疫亲和素，不结合或抑制钙调神经磷酸酶。FK506与Jurkat细胞孵育后，FKBP13和BIG1均有核聚集，但两者未共定位。此外，在缺乏血清的细胞中，BIG1进入细胞核，在核基质和核仁中显示BIG1，并与核孔素有关。在负责的博士后研究员在他的继任者到达之前意外离开造成中断后，对细胞核中与BIG1相关的RNA的表征仍在继续。 对RIpha-BIG2相互作用的研究导致了与去年报道的BIG2分子中A激酶锚定蛋白对应的序列的鉴定。一份准备中的手稿描述了环状AMP对内源性BIG1和其他蛋白质的核积累的依赖蛋白激酶A的作用。探索BIG2与胞外蛋白Exo70(在酵母双杂交系统中检测到)的相互作用导致在反高尔基网络和中心体中以及在沿着微管移动到质膜的胞外结构或复合体中展示了这两种蛋白。所有的发现都与它们在囊泡运输早期和晚期的功能关联一致。

ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate numerous vesicular trafficking pathways. ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs), some of which are inhibited by brefeldin A (BFA), a drug that inhibits protein secretion and causes reversible disintegration of Golgi cisternae. Two BFA-inhibited GEPs (BIG1 and BIG2) that were purified and cloned earler by the group often precipitated together from cultured cells with specific antibodies, but they behaved quite differently in yeast two-hybrid screens, where BIG1 interacted with FKBP13 and BIG2 with RIalpha, a regulatory subunit of cyclic AMP-activated protein kinase (PKA). FKBP13 is an immunophilin of the FK506-binding protein family that does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506 resulted in nuclear accumulation of both FKBP13 and BIG1, but they were not colocalized. In addition, BIG1 moved into nuclei in cells deprived of serum, where it was demonstrated in nuclear matrix and nucleoli as well as associated with nucleoporin. Characterization of RNAs associated with BIG1 in nuclei is continuing after interruption caused by unexpected departure of the responsible post-doctoral fellow before arrival of his replacement. Investigation of the RIalpha-BIG2 interaction led to identification of sequences corresponding to those in A kinase-anchoring proteins in the BIG2 molecules, as reported last year. A manuscript in preparation characterizes protein kinase A-dependent effects of cyclic AMP on nuclear accumulation of endogenous BIG1 and other proteins. Exploring the interaction of BIG2 with exocyst protein Exo70 (detected in the yeast two-hybrid system) led to demonstration of the two proteins in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane. All findings are consistent with their functional association in both early and late stages of vesicular transport.